PROJECT SUMMARY Infectious diseases are an enormous burden on global health, as evidenced by the current COVID-19 pandemic. However, we only poorly understand the many mechanisms that hosts have evolved to defend against viruses and that viruses have counter-evolved to defeat those defenses. Importantly, the result of these host-virus evolutionary conflicts (i.e. whether the host or the virus is successful) ultimately determine human susceptibility to infection and the ability of viruses to zoonotically transmit into the human population. It is therefore of paramount importance that we address the following questions: what are the critical genes and mechanisms that protect us from infection, how do viruses counteract those defenses, and how does host genetic variation affect susceptibility to infection? Our research brings an evolution-guided perspective to answering these questions by exploiting the fact that the interests of viruses and their hosts are necessarily at odds with one another. That is, if the host successfully defends against a viruses, there is evolutionary pressure on the virus to evolve a way to overcome that defense. Likewise, if the virus establishes a successful infection, the host is pressured to adapt. These back and forth dynamics drive constant innovation on both sides of host-virus molecular interactions, resulting in the wide genetic and functional diversity we see today. Our research explicitly leverages this diversity to discover which host proteins have been driven to rapidly evolve by genetic conflicts with viruses, in effect allowing viruses to lead us to the host genes, mechanisms and pathways that are most important for fitness. Based on this evolution-guided approach, our current work focuses on the importance of several incompletely understood post-transcriptional and post-translational regulatory mechanisms in host antiviral defense. One current area of focus is investigating the antiviral mechanisms and evolutionary consequences of a dynamically evolving family of genes, known as IFITs, that distinguish host from viral mRNAs based on mRNA modifications. Another aim is to determine the immune functions of a poorly characterized but rapidly evolving family of genes known as PARPs that catalyze the post-translational addition of ADP-ribose to proteins. Using diverse virology models, coupled with genetic and biochemical approaches, these studies aim to not only determine the consequences of IFIT and PARP gene evolution on susceptibility to viral infection, but also to reveal the broader mechanistic roles for mRNA modifications and ADP-ribosylation in host antiviral defense and cellular regulation. Finally, we are developing genome wide tools to identify other rapidly evolving but understudied regulatory mechanisms that we hypothesize are additional determinants of human susceptibility to viral infection. The overall mission of our work is to use this evolution-guided approach to provide unique insights into mechanisms of host defense and viral immune evasion, species-specific barriers to viral replication that prevent zoonotic transmission into humans, and pathogen-driven evolution of cellular functions.